CCL3 In Plasma Research Articles

Abstract PS03-02: Single-cell metabolic profiling uncovers new precision immunotherapy strategy in triple-negative breast cancer

Abstract Background: Immunotherapy has emerged as a novel cornerstone in the treatment of triple-negative breast cancer (TNBC), while its benefits have been observed in only a limited subset of patients. The intricate metabolic interplay between cancer cells and microenvironmental cells remodels the tumor microenvironment and exerts a significant influence on the responses to immunotherapy. However, there is still a lack of clarity regarding the strategies to target this metabolic crosstalk in order to predict efficacy and enhance sensitivity for immunotherapy. Methods: We conducted single-cell RNA sequencing (scRNA-seq) analysis on a cohort of 27 patients with TNBC receiving either immunotherapy combination regimen or chemotherapy alone. Building upon this foundation, we embarked on a comprehensive investigation of the single-cell metabolic landscape from three distinct perspectives: metabolic genes, metabolic pathways, and metabolic flux, represented by the flux balance analysis (FBA) algorithm Compass. Furthermore, we identified crucial cell subgroups that exhibited a strong correlation with immunotherapy efficacy and elucidated their metabolic characteristics to postulate potential metabolic crosstalk. Then, we employed the in vitro, in vivo, and patient-derived models to unravel the underlying mechanisms of metabolic crosstalk and proposed the translational strategies. Results: Utilizing newly-developed single-cell FBA method, we illustrated that different cell types have distinct metabolic features. Specifically, tumor cells and macrophages had more active metabolic reprogramming and higher metabolic heterogeneity. Furthermore, we illustrated that a subset of tumor cells having active antioxidant metabolism (featured by the upregulation of GSTP1) and macrophage subset depending on glutamine metabolism (characterized by CCL3+) were negatively and positively predictive of immunotherapy responses, respectively. Mechanistically, GSTP1-mediated excessive consumption of glutamine by tumor cells competitively restrained the intake of glutamine into CCL3+ macrophages to promote their ferroptosis and reduce their infiltration. Clinically, we demonstrated that targeting the GSTP1, which is specifically expressed in tumor cells, remodeled immune-metabolic microenvironment and enhanced immunotherapy efficacy. Furthermore, we established the utility of plasma CCL3 abundance, GSTP1 protein abundance, and CCL3 protein abundance, as robust predictors of response to immunotherapy, in several independent immunotherapy cohorts. Conclusion: Our study presented a comprehensive and multidimensional analysis of the single-cell metabolic landscape. Identifying the crucial metabolic crosstalk remodeling the immune microenvironment, our promising finding highlights the potential significance of modulating cell-cell metabolic interactions in sensitizing TNBC to immunotherapy, providing valuable insights for future therapeutic interventions. Citation Format: Ying Xu, Yi Xiao, Han Wang, Zhimin Shao, Yi-Zhou Jiang. Single-cell metabolic profiling uncovers new precision immunotherapy strategy in triple-negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS03-02.

Evaluation of plasma CCL3 levels in individuals with newly diagnosed chronic lymphocytic leukemia and it’s correlation with clinical and laboratory parameters

Abstract: BACKGROUND: The most prevalent type of leukemia in adults is called chronic lymphocytic leukemia (CLL). is a lymphoproliferative condition is defined by the mature clone’s expansion in blood, secondary lymphoid tissues, spleen and the bone marrow. CCL3 is a cytokine that is involved in development, repair, and the acute inflammatory state. CCL3, therefore, should become useful for risk assessment in patients with CLL. OBJECTIVES: The aims of the study were to estimate the level of plasma CCL3 in newly diagnosed patients with CLL matched to healthy controls and correlate it with hematological parameters and clinical parameters and prognostic markers such as CD38 and LAIR-1. PATIENTS, MATERIALS AND METHODS: This study was cross-sectional in nature. Patient group included 55 patients with newly established diagnosis of CLL proven by morphology and immunophenotyping and control group included sex and age matched of 30 healthy individuals. The plasma CCL3 levels were measured using an enzyme-linked immunosorbent assay. Microsoft Office Excel 2019 and SPSS version 26 were used for the statistical analysis. The mean and standard deviation of normally distributed numerical values whereas the terms “median” and “range” were used to describe numerical values that are not regularly distributed. P <0.05 was used to determine the degree of significance. RESULTS: There were statistically significant relationships between CCL3 of patient (median level about 766.42 ng/ml) and control (median level about 453.35 ng/ml) groups (P < 0.001). There was a strong correlation between CCL3 and white blood cell, hemoglobin, and absolute lymphocyte count (P = 0.013, P < 0.001, and P = 0.011, respectively). There was a strong relationship between CCL3 expression with Binet stage and with hepatomegaly (P < 0.001 and P = 0.020, respectively), but no significant relation between CCL3 expression and CD38 and LAIR-1. CONCLUSIONS: Patients with CLL had high levels of CCL3, which increased with advancing Binet stages. Therefore, our research clarified the useful and simplicity of measurement of CCL3 plasma levels for follow-up in CLL patients and risk assessment.

Candidate Biomarkers for Sepsis-Associated Acute Kidney Injury Mechanistic Studies.

Sepsis-associated acute kidney injury (SA-AKI) is a frequent complication of sepsis, yet the pathophysiologic mechanisms of SA-AKI are incompletely understood. PERSEVERE is a clinically validated serum biomarker panel with high sensitivity in predicting mortality from sepsis, and recent evidence suggests it can also predict severe, persistent SA-AKI at day 3 of hospitalization among septic children. We developed a murine model of PERSEVERE (mPERSEVERE) to further interrogate the sepsis-related biological underpinnings of SA-AKI using candidate biomarkers within mPERSEVERE. Eight-week-old C57BL/6 male mice underwent induction of sepsis by cecal ligation and puncture (CLP). mPERSEVERE biomarkers were collected at 8-hours and kidneys were harvested at 24-hours post-CLP Classification and regression tree analysis (CART) was used to generate a SA-AKI predictive model. Kidney gene expression levels of candidate biomarkers were quantified using real time polymerase chain reaction. Thirty- five mice underwent CLP Among mice identified by mPERSEVERE as high-risk for mortality, 70% developed SA-AKI at 24-hours compared to 22% of low-risk mice. CART analysis identified two mPERSEVERE biomarkers-C-C motif chemokine ligand 3 (CCL3) and keratinocyte-derived chemokine (KC)-as most predictive for SA-AKI with an area under the receiver operating curve of 0.90. In mice that developed SA-AKI, renal expression of KC was significantly increased compared to mice without SA-AKI (p = 0.013), whereas no difference was seen in renal expression of CCL3 in mice with SA-AKI vs. no SA-AKI. KC and CCL3 localized to renal tubule epithelial cells as opposed to infiltrating immune cells by immunohistochemistry. The combination of plasma CCL3+KC can predict SA-AKI development in mice at 24-hours following CLP Of these two biomarkers, only renal expression of KC is increased in mice with SA-AKI. Further studies are required to determine if KC directly contributes to the underlying pathobiology of SA-AKI.

Plasma C-C Chemokine Concentrations in Intermediate Age-Related Macular Degeneration.

Purpose: To determine the relationship between plasma concentrations of the C-C chemokines CCL2, CCL3, CCL4, and CCL5 and intermediate age-related macular degeneration (iAMD) patients compared with control inidividuals to further define the inflammatory pathways associated with age-related macular degeneration.Methods: The concentrations of CCL2, CCL3, CCL4, and CCL5 were measured using multiplex assays in plasma collected from 210 patients with iAMD and 102 control individuals with no macular degeneration as defined by multi-modal imaging. Non-inflammatory data included in the analysis were: age, sex, family history of AMD, history of smoking, body mass index, presence of reticular pseudo-drusen and cardiovascular disease. Median concentrations as well as a cutoff value for each chemokine were compared between the two groups.Results: The median concentrations of CCL2 and CCL4 did not differ between control and iAMD groups, however, CCL2 was elevated in iAMD when a cutoff comparison was used (p < 0.05). Median CCL3 and CCL5 concentrations were significantly decreased in the macular degeneration group compared with controls (p < 0.001) as well as when a cutoff value comparison was used. CCL3 and CCL5 were negatively correlated in cases and positively correlated in controls.Conclusions: Plasma CCL3 and CCL5 concentrations were significantly decreased and CCL2 concentrations were increased in patients with iAMD compared with controls, suggesting a role for C-C chemokines in the systemic inflammatory processes associated with disease development.

CXCL13 (BCA-1) Plasma Levels Are a Useful Biological Marker for Disease Progression in Chronic Lymphocytic Leukemia (CLL) Patients

Background: CXCL13 is a chemokine that is constitutively secreted by follicular dendritic cells (FDC) and T follicular helper (Tfh) cells in secondary lymphoid tissues, where CXCL13 plays an essential role in organizing the micro-architecture of germinal centers (GC). Interactions between CLL cells and the lymph node microenvironment mimic, in several aspects, the normal GC reaction. CXCL13 confers access of CXCR5-expressing CLL cells to the lymph node microenvironment. Interestingly, immunization experiments in mice and man demonstrated CXCL13 to be a reliable blood biomarker for GC activity (PNAS 113:2702, 2016). Therefore, we hypothesized that CXCL13 plasma levels reflect disease activity in CLL.Methods: We measured CXCL13 plasma levels by ELISA in a cohort of 400 CLL patients (329 treatment naïve and 71 previously treated) and correlated CXCL13 levels with prognostic markers, time to first treatment (TTFT), and plasma levels of the BCR signaling-related chemokines CCL3 and CCL4 (MIP-1α and β).Results: CXCL13 plasma concentrations were significantly higher in samples from CLL patients with active disease who progressed to require therapy. Accordingly, advance stage CLL patients had higher CXCL13 plasma concentrations than early stage patients. For example, the median CXCL13 concentration in early stage CLL (Rai stage 0, n=120) was significantly lower (80.6 pg/mL, range: 24.4 - 494.4 pg/mL) than in advanced-stage patients (Rai stage III or IV, n=79), median CXCL13: 202.6 pg/mL (range: 32.8 - 1418.0 pg/mL, n=79, p<0.0001). Similarly, median CXCL13 concentrations were significantly higher in patients with high β2 microglobulin, high plasma CCL3 concentrations, elevated LDH, and high white blood counts (WBC). In contrast, the IGHV mutational status, ZAP70, CD38 expression, CCL4 levels and cytogenetic subgroups had little or no impact on CXCL13 levels. Of the 329 treatment-naïve patients studied, 209 eventually required treatment; the median time from sample collected to first treatment (TTFT) for the entire population was 549 days. Patients were dichotomized in high and low CXCL13 levels, based on the approximate median of 128 pg/mL. Based on this, CLL patients with high CXCL13 levels (n=116) had a significantly shorter median TTFT of only 341 days, when compared to patients with low CXCL13 levels (n=93), in whom the TTFT was 758 days (p=0.0019, see Figure). For validation, we analyzed CXCL13 levels in serial plasma samples from patients treated with ibrutinib (n=30). At baseline, prior to treatment, the median CXCL13 levels was 109.6 pg/mL (range: 23.5 - 403.5 pg/mL), and CXCL13 plasma concentrations significantly decreased during treatment to 58.4 pg/mL after 1 week of ibrutinib therapy (20.8 - 159.3 pg/mL, p<0.001). After at 6 months of ibrutinib, CXCL13 concentrations were 42.3 pg/mL (17.6 - 118.2 pg/mL, p<0.001), and at 12 months 35.6 pg/mL (11.5 - 75.5 pg/mL, p<0.001).Conclusion: These studies demonstrate that CXCL13 serves as a novel plasma biomarker for disease activity and progression in CLL patients. Also, these studies emphasize the importance of CXCL13 as a plasma biomarker for B cell activation and expansion in normal or malignant B cell follicles within secondary lymphatic tissues. [Display omitted] DisclosuresWierda:Genentech/Roche: Consultancy, Honoraria, Research Funding; Karyopharm: Research Funding; Genzyme: Consultancy, Honoraria; Gilead: Consultancy, Honoraria, Research Funding; Juno: Research Funding; Kite: Research Funding; Emergent: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria; Janssen: Research Funding; Sanofi: Consultancy, Honoraria; Acerta: Research Funding; GSK/Novartis: Consultancy, Honoraria, Research Funding; The University of Texas MD Anderson Cancer Center: Employment; Pharmacyclics: Consultancy, Honoraria, Research Funding; Merck: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria, Research Funding. Burger:Gilead: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Research Funding; TG Therapeutics: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Research Funding; Novartis: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses; Janssen: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses; Pharmacyclics LLC, an AbbVie Company: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Research Funding.

CCR5/CCR5 ligand-induced myeloid-derived suppressor cells are related to the progression of endometriosis.

Immunological disorders have been reported to promote the progression of endometriosis. Several recent studies have shown that myeloid-derived suppressor cells (MDSC) drive the progression of endometriosis. The aim of this case-control study was to test whether CCR5 and its ligands drive MDSC accumulation and play a role in the progression of endometriosis. Thirty-six endometriosis patients and 20 controls were recruited. All subjects underwent laparoscopy. An ELISA kit was used to define CCR5 ligands in plasma and peritoneal fluid from endometriosis patients; flow cytometry was then used to characterize CCR5+MDSC in peripheral blood and peritoneal fluid. Data showed that endometriosis patients displayed a significantly higher production of plasma CCL3 (P = 0.046) and peritoneal fluid CCL3/5 (P = 0.042/0.036) compared with those from the uterine leiomyoma group. Furthermore, the concentrations of peritoneal fluid CCL5 were elevated in late stage patients compared with those from the uterine leiomyoma group. Accumulation of blood CCR5+Mo-MDSC was detected in endometriosis patients compared with those from both the ovarian dermoid cysts and uterine leiomyoma groups. Endometriosis patients also showed an elevation of CCR5+MDSC and CCR5+Mo-MDSC in peritoneal fluid samples compared with uterine leiomyoma samples. It was also found that enrichment of CCR5+MDSC (r = 0.6807; P < 0.0001) and CCR5+Mo-MDSC (r = 0.6893; P < 0.0001) were correlated with enhanced production of CCL5 in peritoneal fluid from endometriosis patients. This study showed that CCR5 and its ligands could drive the progression of endometriosis by enhancing the accumulation of MDSC. These findings might produce a promising treatment that targets CCR5+MDSC for endometriosis patients.

CCL3 serves as a potential plasma biomarker in knee degeneration (osteoarthritis)

To explore the ability of chemokines in plasma to detect the presence of pre-X-rays defined knee degeneration and the extent (burden). A total of 181 subjects (75 control subjects, 47 pre-X-KD patients and 50 X-KOA patients) were included and subdivided into three subgroups. Articular cartilage loss in pre-X-KD patients were scored on the basis of the ICRS classification during the arthroscopy or documented on MRI with chondral WORMS. The severity of X-KOA was graded using the Kellgren-Lawrence classification through the posterior-anterior knee X-rays. The concentrations of the inflammatory cytokines and chemokines in plasma were quantified using Luminex microbead-based suspension array (SA) and were cross-validated by enzyme-linked immunosorbent assay (ELISA). CCL3 in plasma showed the highest ability to discriminate pre-X-KD patients from the controls with an AUC of 0.799. At a cutoff value of 0.168 pg/ml, the sensitivity was 70.21%, the specificity was 96.00%, the positive predictive value was 91.67% and the negative predictive value was 83.72%. As to define disease burden, the plasma levels of resistin, IL6, IL8, CCL3 and CCL4 showed significant association with the severity of X-rays defined knee OA, with regard to the KL classification. Moreover, significant elevation of IL6, IL8, CCL3 and CCL4 levels in plasma were observed in severe knee OA patients (KL grade IV) compared with those with pre-X-KD (KL grade 0-I). We firstly showed that the plasma CCL3 could be potential serum biomarker for knee OA with the capacity to detect pre-X-rays defined changes and stage the severity of damage in knee.

S47 Enhanced Il-6/ccl3 Signalling In The Plasma Of Patients With Copd

Rationale IL-6 is a pro-inflammatory cytokine that signals through soluble (sIL-6R/sgp80) and membrane bound (gp80) receptors to promote recruitment of mononuclear cells. IL-6 induces expression of CCL3, a monocytic chemokine. Monocytes are precursors of macrophages and dendritic cells. They can be classified into three subtypes according to surface expression of CD14 (LPS receptor) and CD16 (FcgammaRIII): CD14++CD16-, CD14+CD16+, CD14-CD16++. We measured plasma levels of IL-6, sIL-6R and CCL3 and determined the chemokine receptor expression profile of circulating monocytes in COPD. Methods 70 COPD patients and 30 healthy controls comprising 15 smokers (S) and 15 healthy non-smokers (HNS) underwent plasma sampling. Levels of IL-6, sIL-6R and CCL3 were determined by multiplex analysis (MSD) of plasma. Multi-colour flow cytometry was performed on whole blood obtained from 32 COPD patients, 8 S and 8 HNS to measure surface expression levels of chemokine receptors CCR1, CCR2, CCR7, CXCR1 and CX3CR1 on CD14++CD16-, CD14+CD16+ and CD14-CD16++ monocytes. Results COPD patients had the greatest levels of IL-6 and sIL-6R. CCL3 was not detected in any controls, but was present in a subset of COPD patients.% surface expression of the CCL3 receptor CCR1 measured on CD14++CD16- monocytes of COPD patients was greater than those of HNS (p = 0.04). There were no significant differences in expression levels of other chemokine receptors. Conclusion We report evidence of enhanced IL-6 signalling in the plasma of COPD patients and increased plasma CCL3 in a subset of individuals from this disease group. Furthermore, there was increased CCR1 expression on COPD monocytes. Enhanced IL-6 may co-ordinate the mononuclear component of the inflammatory response in COPD.

Target Engagement, Pathway Inhibition, and Efficacy Of The Bruton's Tyrosine Kinase (Btk) Inhibitor CC-292

Target Engagement, Pathway Inhibition, and Efficacy Of The Bruton's Tyrosine Kinase (Btk) Inhibitor CC-292

The AKT Inhibitor MK2206 In Combination With Rituximab and Bendamustine Is Tolerable and Active In Relapsed Or Refractory Chronic Lymphocytic Leukemia: Results From a Phase 1 Study (NCCTG N1087 Alliance)

BackgroundsB cell receptor (BCR) signals and microenvironment protection have been recognized to have key influences on CLL survival and activation. Akt activation is down-stream of the BCR signaling cascade and is critical in the mediation of the bi-directional interactions between CLL B-cells and bone marrow stroma. MK2206 is an allosteric Akt inhibitor previously tested for safety in solid tumor patients. Our preclinical data has demonstrated the synergy of MK2206 with bendamustine (B) to induce CLL apoptosis in vitro. MK2206 also abolished the signal activation and cytokine production induced by BCR ligation in CLL leukemic cells (Ding, ASH 2012). Therefore, we conducted a phase 1 trial testing the safety and efficacy of MK2206 in combination with bendamustine and rituximab (BR) for patients with relapsed/refractory CLL. MethodsA phase 1 dose-escalation study was designed to define the maximum tolerated dose (MTD) as well as the safety and efficacy of 3 dose levels (90 mg or 135 mg or 200 mg weekly) of the oral Akt inhibitor MK2206 in combination with bendamustine (70 mg/m2 daily for 2 days in each cycle) and rituximab (cycle 1: 375 mg/m2, cycle 2-6: 500 mg/m2) therapy in relapse/refractory CLL patients. To test the targeting efficacy of MK2206 on Akt and downstream signals, we designed a one-week “run-in” of single agent MK2206 before the initiation of BR during cycle 1. MK2206 was then given along with BR on day 1 of cycles 2-6. Tumor response was evaluated 2 months after the 6th/last cycle of therapy based on IWCLL 2008 criteria. Correlative laboratory studies included assessment of Akt signaling and the plasma cytokine levels (multiplex beads assay (Invitrogen)) at baseline and after one week of first dose of single agent MK2206. ResultsNine patients (median age 68) were enrolled in the phase I portion of this trial. All 9 patients had unmutated immunoglobulin heavy chain variable region genes (IGHV) and 4 had del(11q). Six had received prior chemoimmunotherapy (CIT: FCR or PCR or PCO) and 5 had high risk disease defined as early progression within 26 months of the last CIT. Three had received alemtuzumab containing regimen. We have observed potent lymphocyte mobilization in all evaluated patients (n=7) after one-dose of MK2206 during week 1 (mean increase ALC: 35%) as well as reductions of plasma CCL3, CCL4 and CCL2 in the majority of the patients. A single dose-limiting toxicity (DLT) (febrile neutropenia and hemolytic anemia) was observed at dose level 1 (90 mg weekly) among 6 patients treated at this dose. Two patients with dose-limiting toxicities at dose level 2 (135 mg weekly) were observed where one patient had a rash and one patient had dehydration and febrile neutropenia among three patients treated at this dose. Accordingly, the 90 mg once weekly dose was determined to be the MTD for phase 2 trial testing. The most frequent grade 3 or 4 adverse events observed to date were neutropenia (44%) including febrile neutropenia (22%), rash (22%), diarrhea (22%), nausea and vomiting (11%), dehydration (11%), hemolytic anemia (11%) and thrombocytopenia (11%). The frequency of cytopenias was similar to the published German experience with BR (50% grade 3 or 4 hematological toxicities compared to 44% in N1087) in relapsed CLL patients (Fischer, JCO 2011). The median number of cycles received was 6 (range 1-6).Using an intent to treat analysis, two patients achieved complete response (CR, n= 2, 22%), one patient achieved nodular partial response (nPR, n=1, 11%) and five patients achieved a partial response (PR, n=5, 56%). The overall response rate (ORR) is 89%. ConclusionThe Akt inhibitor MK2206 in combination with BR is well tolerated in patients with relapsed/refractory CLL. Single dose of MK2206 inhibits Akt phosphorylation, mobilizes leukemic cells and decreases plasma cytokines involved in BCR signals. The preliminary efficacy of this combination demonstrated promising results with a 89% ORR and 22% CR albeit in the small phase 1 cohort. These results compare favorably to BR alone (9% CR and 59% ORR) in relapsed/refractory CLL patients. Phase 2 testing of this combination is now underway. AcknowledgmentThis work was supported by the funding from NCI grant K23CA160345 (WD), NCI grant CA95241 (NEK) and Alliance for Clinical Trials in Oncology (CA025224 and CA33601). Disclosures:Off Label Use: MK2206, used in a phase 1 trial of relapsed CLL disease. Shanafelt:Genentech: Research Funding; Glaxo-Smith-Kline: Research Funding; Cephalon: Research Funding; Hospira: Research Funding; Celgene: Research Funding; Polyphenon E International: Research Funding. Zent:Novartis: Research Funding; GlaxSmithKline: Research Funding; Biothera: Research Funding; Genzyme: Research Funding; Genentech : Research Funding. Reeder:Celgene: Research Funding; Novartis: Research Funding; Millenium: Research Funding.

In Vivo Inhibition of BCR Activation in High-Risk CLL Patients On Therapy with Bruton's Tyrosine Kinase Inhibitor Ibrutinib: Correlative Studies from an Ongoing Phase 2 Clinical Trial

AbstractAbstract 186B cell receptor (BCR) signaling is essential for normal B cell development and plays an important role in several B cell malignancies, including chronic lymphocytic leukemia (CLL). BrutonÕs tyrosine kinase (Btk) transmits B cell receptor (BCR) signaling and can be inhibited by ibrutinib, a selective, covalent Btk inhibitor. Because of highly encouraging results with ibrutinib in high-risk CLL patients in the Phase 1/2 trial, we explored the combination of ibrutinib and rituximab in forty high-risk CLL patients, characterized by the presence of 17p deletion or TP53 mutation (treated or untreated), previously treated patients with 11q deletion, or patients with a short remission duration (< 3 years) after first-line chemo-immunotherapy. Here, we present early correlative studies from this trial, focusing on BCR-related CLL responses (chemokine secretion, viability) and CLL cell migration, based on our recent experience with ibrutinib in preclinical CLL models (Ponader S et al., Blood 119:1182–9, 2012). The aim of this study was to determine if preclinical results of Btk inhibition with ibrutinib can be recapitulated in vivo with specimen from the high-risk population enrolled on this trial. Plasma levels of CLL3 and CCL4 (MIP-1α/β), two chemokines secreted by CLL cells in response to BCR activation, were assessed before, at 14, and at 28 days on treatment with ibrutinib. In 28 analyzed patients, we demonstrate robust, significant reductions in CCL3 and CCL4 plasma concentrations after 14 and 28 days of treatment. As shown in Figure 1, plasma CCL3 levels were reduced from 139.6 (± 40.4) pg/mL before treatment to 6.9 (± 0.9) pg/mL or 6.5 (± 0.7) pg/mL after 14 and 28 days of treatment, respectively. We also evaluated in vitro secretion of CCL3 and CCL4 into supernatants of CLL cells isolated from 12 patients before and during ibrutinib therapy in response to stimulation with anti-IgM. Compared to CLL cells from pre-treatment specimen, CLL cells from patients on Ibrutinib therapy showed reduced levels of CLL3 and CCL4 secretion, and additional treatment with Ibrutinib (0.5 μM – 1μM) led to reduced chemokine levels only in pre-treatment samples, indicating complete Btk target inhibition. We next evaluated the effect of ibrutinib on CLL cell viability after anti-IgM stimulation. In pre-treatment samples, ibrutinib abrogated BCR-triggered CLL cell survival. Surprisingly, CLL cells from ibrutinib-treated patients remained anti-IgM responsive in these viability assays. These Btk-independent pro-survival effects could not be inhibited by in vitro treatment with ibrutinib, indicating that some of the anti-IgM-triggered pro-survival signaling can bypass Btk. Next, we analyzed migration of CLL cells towards the chemokines CXCL12 and CXCL13 in transwell chemotaxis assays. Pre-treatment samples displayed significant higher chemotaxis towards CXCL12 and CXCL13 when compared to CLL samples from patients on therapy with ibrutinib. The mean relative migration of such samples toward CXCL12 or CXCL13 was reduced to 28% (± 5%) or 35% (±15%) of respective CLL cells isolated before ibrutinib therapy (100%), n=6. Collectively, our results demonstrate that Ibrutinib blocks BCR-dependent survival and migration responses in high-risk CLL patients in vivo. They also corroborate the validity and robustness of CCL3 and CCL4 as biomarkers for BCR inhibition in CLL patients. [Display omitted] Disclosures:O'Brien:Pharmacyclics: Research support Other. Buggy:Pharmacyclics: Employment, Equity Ownership. Burger:Pharmacyclics: Consultancy, Research Funding.

Plasma levels of chemokines during leprosy specific treatment

Leprosy, whose etiologic agent is Mycobacterium leprae, is an illness of ample clinical and immunopathological spectrum. Although chemokines seem to be involved in the immunopathogenesis of leprosis, few studies have been carried out to unveil the potential of chemokines as biological markers of the disease. The purpose of this study was to investigate the value of measuring CCL2, CCL3, CCL11 and CCL24 in plasma of patients with leprosy (LE) at different stages of multi-drug therapy (MDT). Chemokines were measured by ELISA in plasma of 30 non-infected individuals (NI) and 33 LE patients before and at different stages of treatment. The plasma concentration of CCL11 ( p < 0.01) and CCL24 ( p < 0.05) was increased in LE patients before treatment when compared to NI individuals. The plasma concentration of CCL24 decreased after MDT ( p < 0.05). No differences were observed in the concentration of CCL2 and CCL3 in plasma of NI and LE individuals. The elevated levels of CCL11 and CCL24 in plasma of patients with LE suggest that these chemokines may play a role in disease pathogenesis. Moreover, the decrease of CCL24 after treatment suggests that this chemokine might be useful as a biomarker of response to MDT in patients with leprosy.

Downregulation of MIP-1α/CCL3 with praziquantel treatment in Schistosoma haematobiumand HIV-1 co-infected individuals in a rural community in Zimbabwe

BackgroundChemokines have been reported to play an important role in granulomatous inflammation during Schistosoma mansoni infection. However there is less information on their role in Schistosoma haematobium infection, or on the effect of concurrent HIV-1 infection, as a potential modifying influence.MethodsTo determine levels of MIP-1α/CCL3 chemokine in plasma of S. haematobium and HIV-1 co-infected and uninfected individuals in a rural black Zimbabwean community.A cohort was established of HIV-1 and schistosomiasis infection and co-infection comprising 379 participants. Outcome measures consisted of HIV-1 and schistosomiasis status and levels of MIP-1α/CCL3 in plasma at baseline and three months post treatment. An association was established between MIP-1α/CCL3 plasma levels with HIV-1 and S. haematobium infections.ResultsA total of 379 adults formed the established cohort comprising 76 (20%) men and 303 (80%) women. Mean age was 33.25, range 17 - 62 years. The median MIP-1α/CCL3 plasma concentration was significantly higher in S. haematobium infected compared with uninfected individuals (p = 0.029). In contrast, there was no difference in the median MIP-1α/CCL3 levels between HIV-1 positive and negative individuals (p = 0.631). MIP-1α/CCL3 concentration in plasma was significantly reduced at three months after treatment with praziquantel (p = 000).ConclusionThe results of our study show that the MIP-1α/CCL3 levels were positively associated with S. haematobium egg counts at baseline but not with HIV-1 infection status. MIP-1α/CCL3 levels were significantly reduced at three months post treatment with praziquantel. We therefore conclude that MIP-1α/CCL3 is produced during infection with S haematobium. S. haematobium infection is associated with increased MIP-1α/CCL3 levels in an egg intensity-dependent manner and treatment of S. haematobium is associated with a reduction in MIP-1α/CCL3.

The role of chemokines in controlling granulomatous inflammation in Schistosoma mansoni infection

Chemokines are a superfamily of low-molecular-weight cytokines that were initially described for their chemoattractant activity. It is now clear chemokines have several other activities that modulate immune processes. Chemokines appear to play a role in the pathogenesis of several inflammatory diseases. The role of chemokines and their receptors in mediating granulomatous inflammation induced by Schistosoma mansoni egg antigens presented in particulate manner have been studied in detail. Much less is known of the role of chemokines in mediating inflammation during the course of S. mansoni infection. Our studies in mice suggest a relevant role for the chemokine CCL3 and the receptor CCR5 in the pathogenesis of experimental S. mansoni infection. Absence of CCL3 is associated with decrease in granuloma size, fibrosis and parasite load. In humans, levels of CCL3 in plasma associate with disease severity and may be useful for diagnostic purposes. In contrast, absence of CCR5 is associated with enhanced lethality, granuloma size and fibrosis. It is suggested that the balance of chemokine production and chemokine receptor activation are important determinants of the fate of infection in experimental animals and humans.